Project Summary/Abstract Dicer is an essential enzyme in all animals, where it cleaves double-stranded RNA (dsRNA) precursors to generate microRNAs (miRNAs) and small interfering RNAs (siRNAs). These small RNAs bind to messenger RNAs to regulate their expression. The proposed studies are focused on Dicer's helicase domain, which is most similar to a family of helicases involved in the innate immune response to viral infection. RIG-I, the founding member of this family, and Dicer, both recognize dsRNA termini. The goal of the proposed research is to understand how Dicer's helicase domain coordinates recognition of its dsRNA substrates, their termini and base-paired structures, to promote conformational changes that correlate with suboptimal (distributive) or optimal (processive) cleavage. Established biochemical assays will be used to perform a comprehensive analysis of Dicer's intrinsic substrate preferences. Features suggested by natural siRNA precursors, such as 5' and 3' overhangs of various lengths, and disruptions to base-pairing such as mismatches and loops, will be monitored for their ability to promote Dicer activities (binding, cleavage, and ATP hydrolysis), and to categorize substrates as optimal or suboptimal. dsRNA substrates that cannot be cleaved by Dicer alone will be assayed in the presence of dsRNA binding proteins (dsRBPs) that facilitate Dicer cleavage. Experiments to reveal the mechanism by which dsRBPs allow termini-independent cleavage will be conducted. The ability to define conditions that lead to distinct conformational states sets the stage for proposed cryo-electron microscopy and x-ray crystallography experiments. These are designed to obtain high- resolution information about the specific substrate interactions involved in the suboptimal and optimal conformations. Studies are designed to fill gaps in knowledge about how Dicer recognizes its naturally occurring substrates, both endogenous dsRNA and exogenous viral dsRNA. The proposed research will create new paradigms for researchers studying Dicer, and those seeking to manipulate Dicer activities for research or therapeutic uses.